Synthesis and Characterization of Biomimetic Hydroxyapatite

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Synthesis and Characterization of Biomimetic Hydroxyapatite Nanoconstruct using Chemical Gradient across Lipid Bilayer Mukund Bahadur Koirala, Tuyen Duong Thanh Nguyen, Arunkumar Pitchaimani, Seong-O Choi, and Santosh Aryal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09042 • Publication Date (Web): 17 Nov 2015 Downloaded from http://pubs.acs.org on November 25, 2015

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ACS Applied Materials & Interfaces

Synthesis and Characterization of Biomimetic Hydroxyapatite Nanoconstruct using Chemical Gradient across Lipid Bilayer Mukund Bahadur Koirala1, 3, Tuyen Duong Thanh Nguyen1, 2, Arunkumar Pitchaimani1, 3, SeongO Choi1, 3, Santosh Aryal1, 2* Nanotechnology Innovation Center of Kansas State1, Department of Chemistry2, Department of Anatomy and Physiology3, Kansas State University, Manhattan, KS 66506

*Corresponding author: [email protected] Department of Chemistry Kansas State University, Manhattan, KS 66506 Tel: +1-785-532-6326

ACS Paragon Plus Environment


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Abstract In

this

study,

we

synthesized

biomimetic hydroxyapatite nanoconstruct

(nanosized

hydroxyapatite, NHAp) using a double emulsion technique combined with a chemical gradient across lipid bilayer for surface modification of a titanium (Ti) implant. The synthesized NHAp was characterized by dynamic light scattering (DLS), X-ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectroscopy, and was further tested for its biocompatibility and in-vitro proliferation efficacy using Normal Human Osteoblasts (NHOst). The results showed that the synthesized NHAp had a hydrodynamic diameter of approximately 200 nm with high aqueous stability. The chemistry of the NHAp was confirmed by FTIR spectroscopic analysis. Typical FTIR vibrational bands corresponding to the phosphate group (PO43-) present in hydroxyapatite (HAp) were observed at 670, 960, and 1000 cm-1. A broad band at 3500 cm-1 confirmed the presence of structural -OH group in the NHAp. Powder X-ray crystallographic diffraction further confirmed the formation of NHAp with characteristic reflections in (002), (211), (130), and (213) planes at respective 2θ degrees. These reflection planes are similar to those of typical HAp crystallized towards (002) and (211) crystallographic planes. The mechanism of the formation of NHAp was studied using the fluorescence resonance energy transfer (FRET) technique. The FRET study showed the fluorescent recovery of a donor fluorophore, the mechanism of the insertion of lipids into nanodroplets obtained from the first water-in-oil (w/o) emulsion during the formation of the second oil-in-water (o/w) emulsion.

With these confirmations, we further studied NHOst cell

proliferation on a Ti surface. When NHOst were cultured on the Ti surface coated with the NHAp, a distinct proliferation pattern and cell-cell communication via cytoplasmic extension on the substrate surface were observed. In contrast, a bare Ti surface showed diminished cell size with minimal adherence. This result indicates that our NHAp covered with a phospholipid bilayer provides a proper environment essential for cell adhesion, which is especially important for bone implants, and the inclusion of NHAp on the Ti substrate would be an effective support for long term sustainability of implants. Key Word: Hydroxyapatite, Lipid, Nanoparticle, Osteoblasts, Implant material


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1. Introduction Calcium in the form of hydroxyapatite (HAp) is a bivalent metal complex, which is one of the major mineral components of the skeletal system. The chemistry of HAp is similar to the inorganic component of bone matrix with a general formula, Ca10(OH)2(PO4)6. Chemical similarities of HAp to natural bone have led to extensive research efforts to use synthetic HAp as a bone substitute and/or replacement in biomedicine either in the form of implant materials or agents for surface modification in order to enhance the biological calcification process of teeth and bone formation.1,2 Biological calcification process is regulated by different biomolecules of the extracellular matrix (ECM) such as collagen, chitin, and proteins. ECM is secreted by cells and performs diverse cellular activities, including sending or receiving instructional information between cells for regulating their growth and terminal cell differentiation, which are highly dependent on the type of cells and the origin of tissue.3,4 One special role of ECM is to provide a template for mineral to deposit in a specific orientation and organization to shape target structure.5,6 The presence of featured minerals of nanoscale, which mimic the nanostructure of bone, on scaffolds and/or implants will synchronize with the ECM, thereby promoting rapid tissue healing and vascularization. Eventually either remnants of the scaffold or implant will be replaced by the new tissue or prolong the functional lifetime of the implant. These complex requirements for the interaction of ECM with bone-mimicking minerals can potentially be met through spatially restricted synthesis of HAp, which can result in the formation of a material with new properties, such as chemical composition, crystallinity, and bio-chemical properties similar to that of natural bone. Although several synthetic methodologies have been investigated and developed for the synthesis of HAp, a new versatile method for HAp synthesis is still of interest due to its immense importance and a wide utilization in biomedical applications as a bone material. HAp is a major component of inorganics of bone composition in the body and shows good biocompatibility and compression strength. Thus, it can be used as infilling materials for the damaged site of bone, artificial ear bones, ocular prosthesis, etc. Various techniques have been developed for generating nanostructured HAp, such as fabricating peptide-amphiphile nanofibers via pHinduced self-assembly and mineralization of HAp,7 generating HAp nanorods through liquid-



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solid-solution technique,8 preparing plate like nanosized HAp via a homogeneous precipitation method in an ultrasound field,9 forming rod-like HAp particles via a biomimetic route,10 synthesizing HAp crystals via urea-assisted hydrothermal method,11 matrix mediated wet chemical synthesis of HAp,12–15 and many others. These synthetic techniques exclusively depend on the properties of scavenging agents such as amphiphilic biomaterials, which control the growth of HAp crystals. No matter what the technique is, the ultimate goal is to develop a nanostructured HAp, which mimics bone composition and is capable of supporting cell growth and proliferation. Considering the components of cell membranes, namely phospholipids, glycolipids, and their supramolecular assemblies as a universal modality of a living system, the utilization of this system could play a vital role in inducing a novel structure of interest. However, less attention has been given to these structural components as a matrix for the synthesis of HAp, which could prolong the functional durability of implant. For example, the ECM of all tissues is composed of phospholipids, lipoproteins, collagen, chitin, and proteins, which govern protein folding, bioactivity, and protein-protein interactions while bone remodeling. The binding and release of growth factors to/from hydrophilic or hydrophobic pockets of the ECM proteins mediate their activity and localization, which are critical for tissue maintenance and regeneration.16 To this end, we adopted a chemical gradient across the lipid bilayer to synthesize a new coreshell nanoparticle (NP) consisting of an inorganic HAp as the core and phospholipids bilayer as the shell. This nanostructure is a construct made up of phospholipid and phospholipid conjugated polyethylene glycol (lipid-PEG) with an interior of nano-crystalline HAp, herein it is called nanosized hydroxyapatite (NHAp). We envision that coating of this NHAp would enhance the absorption of ECM proteins due to the phospholipids on the surface of NHAp, which provide an exultant environment for cell growth by mimicking cell surface. Also, this nanostructure could be used for the surface modification of titanium implants to enhance biocompatibility and proliferation of osteoblast cells.

2. Materials and Method 2.1. Materials


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(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene (ammonium

salt)

(DSPE-PEG-COOH),

glycol)-2000]

1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-

glycerol) (sodium salt) (DSPG), L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Egg-Transphosphatidylated, Chicken) (PE-RhB), (1,2-distearoyl-snglycero-3-phospho ethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (DSPENBD), were purchased from Avanti Polar Lipid Inc (Alabaster, AL, USA). Normal Human Osteoblasts (NHOst) were purchased from Lonza Group Ltd. (Allendale, NJ, USA) and maintained according to the manufacturer’s recommendation. Titanium foil of thickness 0.5 mm was purchased from Fisher Scientific and used as a model implant material. All other chemicals and solvents were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and used as received. 2.2. Synthesis of Nanosized Hydroxyapatite (NHAp) NHAp was synthesized by water-in-oil-in-water (w/o/w) double-emulsion solvent evaporation technique followed by gradient diffusion of disodium hydrogen phosphate. First, an aqueous solution containing calcium chloride was emulsified with a solution of phospholipid in chloroform. Resulting w/o first emulsion is the droplet of Ca2+ anchored with the glycol head moiety of the phospholipid DSPG. Immediately, the first emulsion was further emulsified with an aqueous solution of phospholipid (DSPG) and phospholipid-PEG (DSPE-PEG-COOH) to form second emulsion (o/w). Under these w/o/w circumstances, nanoparticles (NPs) formed were stabilized by PEG and surface lipid bilayer. The interior of the particle contains calcium chloride. The synthesis protocol was optimized to obtain homogeneous and well-dispersed spherical NPs. The resulting NPs were purified and redispersed into the aqueous solution of disodium hydrogen phosphate while heating at 50 °C for 2 h. This helps to diffuse sodium phosphate into the core of NPs, where it reacts with calcium chloride resulting in the formation of calcium phosphate nanocrystals. In a typical experiment, the first emulsion (w/o) was prepared by probe sonication of mixture of 40 µL (250 mM CaCl2) and 1 mg of DSPG in 5 mL of chloroform. Within 2 minutes of sonication under ice-cold conditions, a thick white emulsion was formed. In this first emulsion, an aqueous nano-droplet containing Ca2+ was stabilized by DSPG. Immediately after the formation of the first emulsion, it was further emulsified with a mixture containing 1 mg of DSPE-PEG-COOH and 0.1 mg of DSPG in 10 mL of water (o/w second emulsion) under probe sonication for 5 minutes resulting in the formation of w/o/w



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double emulsion. This mixture was kept under stirring overnight to evaporate chloroform from the emulsion. A care was taken to avoid the solvents separating into two layers, which could possibly destroy the emulsion resulting in the disruption of nanoconstructs. After complete evaporation of chloroform, NPs formed were washed with deionized water using Amicon® ultra centrifugal filter (Mw cut off = 10 kDa) to remove any released calcium chloride during the process. Following the purification, nanoparticles were emerged into a beaker containing 10 mL of 250 mM Na2HPO4 solution. This mixture was allowed to stand for 3 hours at 50°C to diffuse phosphate ions into the core via the lipid bilayer, where mineralization of HAp takes place. The formed NHAp was then purified using Amicon® filter and was kept at 4°C for further use. The various steps involved in the synthesis of NHAp are schematically represented in Figure 1. 2.3. Characterization of NHAp Size and zeta potential measurements of NHAp were carried by dynamic light scattering (DLS) using a Zetasizer Nano ZS apparatus (Malvern, Worcestershire, UK). NHAps were suspended in deionized water at a concentration of 0.1 mg/mL. The Smoluchowski model was used to calculate the zeta potential value. All data represent average of triplicate measurements of samples prepared in three different preparations. The structure of NHAp was further studied by a Transmission Electron Microscope (TEM, Tecnia G2, Sprit Bio TWIN)) operated at 120 KeV. TEM images were analyzed by GATAN digital imaging system (GATAN, Inc.). TEM samples were prepared by drop casting and evaporation technique using fomvar coated cupper grid (400 mesh). Elemental composition was acquired using energy dispersive X-ray analysis (EDX, Oxford Instruments). Fourier transform infrared spectroscopy (FTIR, Cary 630, Agilent Technologies) and powder X-ray Diffraction spectroscopy (D8 AVANCE, Bruker AXS) were used to study the chemistry and crystallographic structure of NHAp, respectively. Scanning electron microscopic (SEM) images were acquired to study the morphology and cellular proliferation using Hitachi S-3500N scanning electron microscope at an operating voltage of 20 keV. 2.4. Fluorescence Resonance Energy Transfer (FRET) Study Formation of stable double emulsion with uniform size distribution via insertion of lipids from second emulsion was studied using the FRET experiment. The fusion of lipids to the nanodroplet from first emulsion was conducted by following a previously described protocol based on


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fluorescence resonance energy transfer (FRET) technique.17,18 In a typical experiment, a fluorescence donor (0.1 mol% DSPE-NBD, excitation/emission = 470/525 nm) and a fluorescence acceptor (0.7 mol% PE-RhB, excitation/emission = 550/590 nm) were incorporated into the droplet of first emulsion. In this composition, the fluorescence emission from the donor was maximally quenched by the acceptor. Then the FRET pair-loaded first emulsion were mixed with a lipid mixture of second emulsion. The fluorescence emission spectra at the range of 500 to 700 nm were obtained with an excitation at 470 nm. All measurements were carried out at 25 °C using a fluorescent spectrophotometer (Synergy H1 Hybrid Reader, BioTek). 2.5. In-vitro Cytotoxicity In-vitro cytotoxicity of NHAp was evaluated using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide) assay against NHOst. In brief, cells were seeded in a 96-well plate at 10,000 cells per well in a complete medium and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C for 24 h. To determine the effect of NHAp on cell viability and proliferation, cells were treated with NHAp suspended in a complete medium at different concentrations (7, 12.5, 25, 50,100 and 150 µg/mL). Cells were further incubated for 24 h. After incubation, MTT working solution was added according to the manufacturer’s instructions. The absorbance was measured at 570 nm in a microplate reader (SynergyH4, Bioteck, Winooski, VT), and the data were collected. Control NHOst cells were maintained without NHAp treatment. The data represents mean ± standard deviation (n=6). 2.6. Titanium Surface Modification and Protein absorption Study Prior to cellular proliferation experiments, titanium (Ti) foil (cut into 2.5×2.5 mm pieces) was cleaned by acetone followed by deionized water three times. This process helps to clean and remove organic contaminants and dust on the surface of the Ti substrate. The obtained clean Ti surface was then subjected to heat treatment at 250°C in an oven overnight in an atmospheric condition. The glossy Ti surface turned a little pale due to titanium oxide formation on the surface. This heating process also helped to sterilize the surface and formed a biocompatible thin oxide layer. With the formation of an oxide layer on the Ti surface, the substrate is maintained under the clean environment. The sterile substrate surface was further modified with NHAp coating by a drop casting and drying method. The NHAp treated Ti surface was gently rinsed with phosphate buffered saline (PBS) to remove any excess of NHAp. The morphology of bare



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Ti, lipid only coated Ti, and NHAp coated was studied using SEM. In a typical experiment, substrate was coated with palladium using sputter coating and the images were acquired at 20kV operating voltage with 10K magnification in order to visualize surface micro structure on Ti. Protein absorption onto the bare Ti, lipid coated Ti, and NHAp coated was studied using Bovine Serum Albumin (BSA) as a model protein. In our experiment, each piece of different Ti substrate in a 96 well plate was incubated with calculated amount of protein for 24 h in a cell culture CO2 incubator mimicking cell culture environment. After 24 h, substrate were selectively removed from the wells using fine tweezer. Substrates were gently rinsed with 100 µL phosphate buffered saline (PBS) in order to remove any unabsorbed protein. Absorbed protein onto the Ti substrates was detached via sonication in 100 µL PBS and analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV-spectrophotometer to determining optical density (OD) at 208 nm, which is the characteristic maximum absorption wavelength for BSA. 2.7. Cellular Proliferation For cell culture experiment, NHOst cells from passages 3−4 were cultured in a T-25 cell culture flask using OGM Bullet Kit™ (Lonza Group Ltd.) supplemented with 10% FBS and 1% antibiotics at 37 °C in a humidified 5% CO2 incubator. During each passage, NHOst cells were treated with 1 mL of 0.1% Trypsin for 3 to 5 min to detach them from the culture flask. Detached cells were treated with 2 mL of trypsin neutralizer and diluted to the required calculated volume with cell culture medium for calculating the cell numbers or to be used in further experiments. The maintained cells were cultured in a 96 well plate (10,000 cells per well) in the presence and absence of the surface modified Ti substrate to study cellular proliferation. Three types of Ti substrate was used in this study namely (1) bare Ti, (2) lipid only coated Ti, and (3) NHAp coated Ti. 3. Results and Discussion HAp is the main component of bone, which possesses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues. Living bone constantly undergoes a coupled resorptive-formative process known as bone remodeling. The process involves simultaneous bone removal and replacement through the respective activities of osteoblasts and


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osteoclasts with the accompanying vascular supply and a network of canaliculi and lacunae. Bone is a complex hierarchical structure and its formation and maintenance are governed by cellular events.19 Implant surfaces with nanostructures superimposed on a micron scale topography mimic this structural hierarchy of bone formation. During bone formation process, osteoblasts produce tropocollagen,20 which self-assembles into an extracellular matrix of collagen fibrils.21 Subsequent mineralization causes the osteoblasts to become entrapped within the bone matrix and differentiate into osteocytes.22 The alignment of the osteocyte lacunocanalicular network reflects the pattern of extracellular matrix formation (i.e., osteocyte lacunae are aligned parallel to the lamellar direction while the canaliculi extend perpendicularly),23 and could be used to interpret the formation and remodeling processes. Considering the aforementioned complex events for bone remodeling and the topographical mimicry (i.e., phospholipid bilayers with the interior containing bone ingredient mineral calcium phosphate), the NHAp proposed herein favors the osteoblast proliferation and cell-cell communication. Cellular proliferation and communication are the preliminary steps for the bone remodeling process. Under this ground, we hypothesized that NHAp surrounded by phospholipid bilayer resembles a cell structure so that it can easily fuse with cell surface, absorb mineral containing Ca2+, and enhance the proliferation of osteoblasts in a way similar to that occurs in in-vivo environment. 3.1. Synthesis and Characterization of NHAp NHAp fabricated using the w/o/w and gradient method was characterized for their physicochemical properties, including size, polydispersity index (PdI), morphology, and surface electrostatic charge. As demonstrated in Figure 2A by dynamic light scattering (DLS) analysis, the NHAp exhibits a uniform size of 200 ± 10.4 nm in diameter with a narrow PdI of 0.180 ± 0.053, suggesting the formation of monodispersed nanoparticles (Figure 2A). The surface zeta potential of NHAp was determined to be −25 ± 6.3 mV (Figure 2B). The negative zeta potential is due to the end carboxylate group of the PEG moiety, which helps the steric stabilization of the NHAp. The NHAp’s morphology under TEM (Figure 2 C& D) showed monodispersed spherical structures of 150 nm in diameter with no sign of bulk aggregation. The difference in DLS and TEM size is due to the fact that DLS exhibit the hydrodynamic diameter whereas TEM is the dry state observation at high vacuum environment. The interior of NHAp is loaded with



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the crystals of HAp with Ca:P ratio of 1.56, which is nearly equivalent to that of naturally occurring HAp. 3.2. Chemistry of NHAp FTIR spectra of the NHAp lyophilized powder show numerous sharp peaks between 670 and 1200 cm-1, which correspond to various PO43- vibrational modes: υ2 (1000 cm-1), υ4 (670 cm-1), and υ1 (960 cm-1), respectively (Figure 3A). The spectra also show adsorbed water (broad peak 2700−3500 cm-1) as well as bands associated with carbon−oxygen bonds. These carbon−oxygen bands can be attributed to the -CH2-CH2-O- moiety of PEG, wherein bands at 3000 and 3500 cm1

are attributed to the adsorbed water (Figure 3A). Finally, the droad peak seen at 3500 cm-1 as

well as the weak peak observed at 700 cm-1 may be attributed to -OH stretch, which is the indicator of the formation of the characteristic apatite-like structure.24 Similarly, characteristic – NH bending at 1450 cm-1, carbonyl stretching at 1750 cm-1, and C-N bending at 750 cm-1 reflect the existing properties of lipids used in the synthesis of NHAp. In addition to the study of functionality of NHAp via FTIR, the crystallographic study was also performed using powder xray diffraction spectroscopy (Figure 3B). All diffraction peaks of NHAp could be identified as hydroxyapatite (JCPDS Reference code: 00-009-0432). The shape of the sharp diffraction peaks indicated that the fabricated hydroxyapatite was fairly well-crystallized, which is presumably due to the crystal growth induced by calcium nuclei into the interior of the nanoparticle. The sharp and intense (002) and (211) reflection planes indicated that hydroxyapatite crystals preferred to be aligned apparently along the c-axis of HAp. This is also evident from the energy dispersive Xray (EDX) analysis of elements, which shows Ca:P ratio of 1.56. This ratio is nearly equivalent to the Ca:P ratio of hydroxyapatite, 1.67. Further, to understand the crystallographic similarities of NHAp to that reported in JCPDS data, we prepared the calcium phosphate in bulk by simple precipitation reaction between calcium chloride and disodium hydrogen phosphate. Crystallographic reflection of bulk-prepared calcium phosphate shows three prominent reflection at 2θ degree 11.6, 21, and 29, which is the characteristic feature of dicalcium phosphate dehydrated (DCPD).25 Major reflection of HAp towards (002) and (211) planes are lacking in case of bulk precipitation, which can be attributed to the fact that mineralization kinetics in case of double emulsion is a well-controlled system as that of simple precipitation . Therefore, the


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chemistry of NHAp is similar to the composition of Ca and P found in naturally occurring bone, which is 1.67. 3.3. Surface morphology and Protein absorption SEM images and elemental composition of Ti foil, lipid vesicles without HAp, and NHAp coated Ti substrate were presented in supporting information Figure S1. Significant differences can be seen in all micrographs, where irregularly roughened at microscale with small pits and cracks in Ti foil (Supporting information Figure S1 A) where filled with lipid (Supporting information Figure S1 B) or NHAp (Supporting information Figure S1 C) coatings. In which NHAp coated surface shows particulate aggregates whereas lipid only coated micrographs shows bulk irregular film of lipids onto the surface of Ti. The qualitative increased roughness of NHAp coated Ti as that of lipid coated Ti is probably due to the fact that these nanostructures are highly stable with solid interior core, which finally forms the clusters on the surface of Ti providing the a qualitative roughness and mineral nutrients for cell proliferation. In addition, all pits and cracks have been filled after coating that could be helpful for adhesion of cell onto the substrate. The different surface microtopographies are known to modulate bone cell differentiation and mineralization on titanium implant materials.26 Therefore, the roughness of the NHAp coated Ti might promote the high activity of the osteoblast attachment as compare to that of lipid only coated Ti substrate. Moreover, surface topography and chemistry highly alter the protein absorption on to the substrate. To this end, we have conducted protein absorption study using BSA as a model protein on three different substrate and the results were presented in Figure 4. As compare to bare Ti and lipid coated Ti, the NHAp coated shows the higher degree of protein absorption (Figure 4A). This absorption study was conducted by incubating calculated amount of protein with piece of each substrate under cell culture environment. After the completion of incubation, substrates were removed and the total protein absorbs were calculated to determine the absorbed protein. On the other hand, absorbed protein in the individual substrates were analyzed using SDS-PAGE electrophoresis. In all type of substrates, absorption of protein was confirmed by visualizing the characteristic band belongs to that of BSA (Figure 4B). The quantification of each band was also performed using imageJ software and the data was presented in the supporting information Figure S2, which shows that amount of protein absorb onto the substrate is higher when surface was modified by NHAp.



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3.4. Mechanism of the Formation of NHAp. The possible mechanism behind the formation of NHAp is presented in Figure 5. It is probably composed of three stages: nucleation, seed formation, and growth. The first stage is the nucleation process, which is the initial reaction between Ca2+ and the glycol moiety of DSPG to generate calcium nuclei. Second step is the formation of hydroxyapatite seed in the ionic sea of Ca2+ and (PO4)3- ions. The formation of HAp seed takes at the interior of the vesicle due to the migration of phosphate ions across lipid bilayer in the direction of lower concentration. Movement from a high concentration to a low concentration is also referred to as movement “with” or “in the direction of” the concentration gradient or “downhill.” When phosphate interact with Ca++ at the interior of the vesicles induce the nucleation of HAp, which in turn reduces the soluble

phosphate

concentration

at

the

interior,

thereby

creating

an

environment

thermodynamically favorable to migrate phosphate ions inside the vesicles until it reaches to the equilibrium. This process is further facilitate efficiently at mild temperature as the mobility of ions increases. The HAp seeds finally grow to form HAp crystals at the core of NPs. The nucleation process is often driven by thermodynamic parameters related to the particle size, which is restricted by the interior size of the NHAp as the crystal grows into the aqueous core of NPs. During the second stage, HAp crystals are generated from the growth of nuclei, which is determined by the surface energy that must be minimal for a given volume for a crystal in equilibrium with its surroundings. Obviously, the surface atoms have fewer nearest-neighboring atoms, compared with bulk atoms, which will raise the energy and therefore lower the stability of those atoms as well as the surfaces themselves.

27

In order to minimize the surface energy,

directed bonds in anisotropic lattices raise crystallization in rods or platelets, leading to the formation of one- or two-dimensional particles such as cubes, rods or plates.28 In present scenario the surface to volume ratio is large, because the reaction occurs in the nano-aqueous core of the nanoparticle thereby resulting in the formation of HAp nano-crystallites inside the core of NPs.

Next, we studied the mechanism of nano-emulsion formation. As can be seen from the proposed mechanism, the first emulsion process is straight forward; the aqueous droplets are being anchored by a self-assembled hydrophilic head moiety of DSPG containing glycol chelator. These w/o emulsion droplets are surrounded by hydrophobic tails of phospholipid embedded in


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the organic phase. However, the structure of second oil-in-water (o/w) emulsion is different from first emulsion as the bulk water phase contains the DSPE-PEG-COOH stabilizer. In order to understand the actual orientation of DSPG-PEG-COOH and how it helps to stabilize the particles from the first emulsion, we performed fluorescence resonance energy transfer (FRET) experiment where a FRET pair of fluorophores, which are covalently attached to the hydrophilic head moiety of the phospholipid, were loaded into the particles while forming the first emulsion. FRET is a widely used technique that measures the distance of two molecules at the molecular level based on an energy transfer mechanism between two fluorophores.17,29 Here, we incorporated a fluorescence donor (DSPE-NBD) and a fluorescence acceptor (PE-RhB) into the membrane of the first emulsion droplet. When the two fluorophores are in close proximity, the excited donor fluorophore can transfer energy to the acceptor fluorophore through a nonradiative long-range dipole–dipole coupling mechanism.17,29 By controlling the molar ratio between the donor and the acceptor (1:7), we prepared FRET-labelled first emulsion in which the fluorescence emission from the donor was maximally quenched by the acceptor as demonstrated in Figure 5B (spectrum in solid line, λem 590 nm). This FRET effect and its efficiency would be reduced as the distance between the donor and the acceptor becomes longer resulting in fluorescence recovery of the donor fluorophore (Figure 5, spectrum in dotted line).17,18,29 As shown in Figure 5B, after the formation of the second emulsion, the purified NHAp was subjected to the fluorescence study and the significant fluorescence recovery was observed at 525 nm, which is the characteristic fluorescence emission peak of DSPE-NBD. The fluorescence recovery of donor is only possible when DSPE-PEG-COOH is fused and inserted into the nanoparticle from the first emulsion. This further confirms the formation of monodispersed NHAp without any lipid micelles in the system, supporting the results form dynamic light scattering that showed monodispersed NPs with narrow PDI.

3.5. In-Vitro Cytotoxicity and Cellular Proliferation. Prior to cellular proliferation experiments, the biocompatibility of NHAp was tested by cytotoxic evaluation against Normal Human Osteoblast cells (NHOst) at various concentrations of NHAp using the MTT assay. Figure 6A shows cell viability over a wide range of tested NHAp concentrations. Even at the highest concentrations of NHAp tested (150µg/mL), NHOst cells were remained viable for 24 h and the cell viability was not affected by the presence of NHAp



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compared to the untreated control cells. This result suggests that NHAp is highly biocompatible with NHOst cells. With this assurance, we next performed the cellular proliferation study onto the titanium surface with and without NHAp coatings.

Surface properties of an implant material have a critical role in cell adhesion process. Osteoblasts cell adhesion on an implant material surface is essential for the success of any implant in which osseointegration is required.30 Figure 6 (B, C, and D) shows representative SEM images of fixed cells on the surface of Ti substrate without (Figure 6B) and with (Figure 6 C & D) NHAp modification. In addition, lipid only coated Ti substrate was also used as a control to culture cell and the SEM micrographic result showing cellular growth was presented in supporting information Figure S3. A clear cellular cytoplasmic extensions appear to be preferentially attached to the surface of the NHAp modified Ti surface as indicated by the arrows in the images. In contrast, cells on the surface of a bare titanium surface don’t show such events of cellular proliferation; cells were contracted and cellular destruction was observed (Figure 6 B). Cells on to the lipid coated Ti show cellular adhesion however the degree of substrate adhesion is not as prominent as that of NHAp coated surface. When the surface is modified with NHAp containing phospholipids and hydroxyapatite nano-crystallites that mimic the natural bone and its surrounding protein environment, cellular communication was observed. In Figure 6D, intercellular communication through cytoplasmic elongation is clearly visible. The possible cellular communication mechanism is presented in the schematic representation (Figure 6 E), where cell’s cytoplasmic elongation adheres to the NHAp aggregates on the Ti surface. The interaction between the NHAp and the cells ultimately leads to the fusion of phospholipids and cellular membrane to be a part of cells in a manner possibly similar to that demonstrated in our lipid insertion experiments (Figure 5). This fusion process increases the mineral calcium concentration in the vicinity of cells. This phenomenon is probably coupled with the secretion of cytokine for communication with neighboring cells.31 In such a situation, slightly elevated Ca2+ concentrations may be helpful to stimulate the proliferation activities of osteoblasts and depress osteoclast-mediated bone resorption via negative feedback loops32. Furthermore, much higher Ca2+ concentrations promote osteoblast differentiation leading to bone mineralization33. Considering the aforementioned phenomena for osteoblast proliferation, the current synthetic


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NHAp system is in favor of osteoblast proliferation as higher Ca2+ is expected around the cellular microenvironment.

4. Conclusions

The synthesized NHAp nanoparticles exhibit excellent biocompatibility and provide the cellular proliferative environment when deposited on the surface of Ti foil, which was used as a model implant material. NHAp was synthesized by a w/o/w double emulsion technique combined with the chemical gradient method across lipid bilayer. Considering the chemistry and crystallographic alignment of the NHAp, which are similar to those of naturally occurring HAp, the superiority of our chemical gradient-based synthesis strategy that uses phospholipids as a building block. The synthesized NHAp possessed hierarchical nanostructures in the interior, which is filled with nano-crystallites of hydroxyapatite and were highly uniform. The results suggest that the inclusion of naturally occurring phospholipids might be one of the critical factors to be considered in the design of new bone implant biomaterials.

5. Acknowledgments The authors acknowledge support from the Department of Chemistry and the Nanotechnology Innovation Center of Kansas State (NICKS), Kansas State University (KSU), Manhattan, Kansas.



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Figure 1. Schematic representation of the colloidal procedure for the synthesis of nanosized hydroxyapatite (NHAp). Emulsion I (w/o) is the CaCl2 aqueous droplet stabilized by oil phase containing DSPG. Emulsion II (o/w) is prepared by introducing the first emulsion into an aqueous solution (blue color) containing DSPG and DSPE-PEG, which stabilizes nanoparticles. Finally, the nanoparticles formed were immersed in the aqueous solution rich in PO34-, resulting in the formation of NHAp.


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Figure 2. Physiochemical characterization of nanosized hydroxyapatite (NHAp). (A) Dynamic light scattering measurement of NHAp showing its hydrodynamic diameter, (B) Surface zeta potential of NHAp, (C) Transmission electron microscopic (TEM) micrograph of NHAp showing monodispersed nanoparticles, and (D) High resolution TEM micrograph of a single NHAp nanoparticle showing the clear anatomical structure containing HAp nano-crystallites at the core.



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Figure 3. Chemistry of NHAp. (A) FTIR-ATR spectrum taken from lyophilized powder of NHAp, (B) X-ray crystallographic reflection of NHAp in comparison with that of calcium phosphate prepared in bulk by simple precipitation reaction between CaCl2 and Na2HPO4, and (C) Study of calcium and phosphate ratio using energy dispersive x-ray (EDX) analysis.


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Figure 4. Protein absorption study performed onto the different Ti substrates viz.; bare Ti, Lipid coated Ti, and NHAp coated Ti substrates incubated with BSA for 24 h in cell culture environment. After incubation, substrates were collected and gently rinsed with PBS to remove any on absorbed BSA. (A) Absorption study conducted on the supernatant after detaching BSA from substrate was studied at 280 nm, characteristic higher absorption wavelength for BSA. (B) SDS-PAGE analysis of absorbed BSA onto the different substrates.



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Figure 5. Study of possible mechanism of the formation of nanosized hydroxyapatite (NHAp). (A) Schematic representation showing each possible step of the formation of NHAp. (B) Fluorescent resonance energy transfer (FRET) mechanism showing the insertion of DSPE-PEG during the second oil-in-water (o/w) emulsification process. Dotted line (_

_ _

) represents the

spectrum where the fluorescence intensity of donor is maximally quenched and solid line (___) represents the insertion of lipid into the first emulsion resulting the recovery of donor fluorescence intensity. DSPE-NBD (λem = 525 nm, donor fluorophore) and PE-RhB (λem = 590, acceptor fluorophore) at molar ratio of 1:7were excited at same wave length (λex = 470 nm). Recovery of the donor fluorescence at 525 nm is due to the increased distance between the donor and acceptor, indicating the insertion of DSPE-PEG into the nanoparticle.


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Figure 6. Cell viability and proliferation study. (A) In-vitro cytotoxicity (MTT assay) of NHAp at various concentrations against Normal Human Osteoblast cells (NHOst). (B) Proliferation of NHOst on a bare Ti substrate without surface modification, (C) NHOst cell cultured on the Ti surface modified with NHAp. Arrows indicate the cell-substratum interaction and adhesion. (D) Cellular communication via cytoplasmic elongation on the NHAp modified Ti substrate. (E) Conceptual schematic of cellular proliferation and communication via fusion of cells with aggregates of NHAp on the NHAp modified Ti surface.



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Table of Contents (TOC) Graphic


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Synthesis and Characterization of Biomimetic Hydroxyapatite

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